EP0291594B1

EP0291594B1 - Pyrazine for use in the treatment of haemodynamic and metabolic disorders

Info

Publication number: EP0291594B1
Application number: EP87304503A
Authority: EP
Inventors: Eiichi Hayashi; Noriyasu Takayanagi; Katsumi Nishimura; Masao Yaso; Yukio Suzuki
Original assignee: Toyo Jozo KK
Current assignee: Asahi Kasei Corp
Priority date: 1986-02-20
Filing date: 1987-05-20
Publication date: 1991-08-07
Anticipated expiration: 2007-05-20
Also published as: EP0291594A1; JPS62195328A; ES2039436T3; DE3772047D1; US4721713A

Description

This invention relates to an agent for improving haemodynamic and metabolic disordes.
Recently, there have been reports on compounds having an inhibitory effect on platelet aggregation. Among these compounds, ones having a pyrazine, ring as a main nucleus have not been much reported. Only the compound tetramethylpyrazine [Abst. of the 16th congress of heterocyclic chemistry (Osaka) p. 65-68 (1984)] and 2-higher fatty acyloxymethyl pyrazines (JP-A-59-219269) are known.
We have synthesised various kinds of pyrazine derivatives and have screened their pharmacological activities. We have found that 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine indicated an inhibitory effect on platelet aggregation, increasing effect on blood flow for organs and tissues and/or protective effect against anoxia which reveal advantageous pharmacological activities. 2-Hydroxy-3-isopropyl-5,6-dimethylpyrazine is a known compound. However pharmacological activity-thereof has not been known.
J. Am. Chem. Soc., 74:1580-1584(1952), G.Karmas et al, describes the preparation of various hydroxypyrazines from ∝-dicarbonyl compounds and hydrohalides of aminoacid amides. 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine is described, but no possible therapeutic activity is mentioned.
US-A-3720768 discloses that aspergillic acid and some derivatives thereof can be used to treat hypertension. However the derivatives described do not include 2-hydroxy-3-isoproyl-5,6-dimethylpyrazine.
The present invention provides 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or a non-toxic salt thereof for use in a method of treatment of the human or animal body by therapy. The compounds may be used in the treatment of haemodynamic and metabolic disorders. For example, they may be used in inhibiting platelet aggregation, as a vasodilator and in protecting against anoxia. The preferred salt is the hydrochloride.
The invention further provides the use of 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or a non-toxic salt thereof in the preparation of a pharmaceutical composition for use in a method of treatment of the human or animal body by therapy, especially for use in a method of treatment of a haemodynamic or metabolic disorder. Also provided are pharmaceutical compositions comprising 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or a non-toxic salt thereof as active ingredient and a pharmaceutically acceptable carrier or diluent, and a process for the preparation of a pharmaceutical composition, which process comprises mixing 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or a non-toxic salt thereof with a pharmaceutically acceptable carrier or diluent, the said mixing being effected under sterile conditions and/or the obtained mixture being sterilised.
Also part of the invention of non-toxic salts of 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine. These are prepared by a process comprising condensing 2,3-butanedione with valineamide in the presence of a tertiary amine and converting the resultant 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine into a non-toxic salt thereof.
Examples of non-toxic salts are pharmacologically acceptable salts, for example inorganic or organic salts such as the hydrochloride, sulfate, carbonate, nitrate, hydrobromide, phosphate, sulfonate, acetate, oxalate, tartrate, citrate, malate, glutamate or aspartate.
The 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or non-toxic salt thereof can be a hydrate. The hydrate is also included in the present invention.
The 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine can be produced according to the general principles of the method disclosed in Reuben G. Jones; J. Am. Chem. Soc., 71: 78-81 (1949) , namely by condensing 2,3-butanedione with valineamide, a type of α-amino acidamide in the presence of a tertiary amine such as triethylamine.
The pharmacological activities and toxicities of 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine hydrochloride are now illustrated.

Test Example 1

Inhibitory effect on platelet aggregation:

(1) Effect in vitro :

Solution of agents to be tested (final concentration 100 µM) were added to platelet rich rabbit plasma containing 3.8% sodium citrate (1/10 v/v) and incubated at 37 °C for 3 minutes. An aggregating agent, adenosine-5-diphosphate (final concentration 2.5µM), was added thereto and the platelet aggregating potency was measured by an aggregometer.
The result is shown in Table 1. The compound used in the present invention shows a distinct inhibitory effect on platelet aggregation. However control agents, tetramethylpyrazine, ticlopidine and acetyl salicyclic acid have not inhibitory effect.

(2) Effect in vivo:

The agents to be tested were administered orally to male Wistar rats, 5 animals in a group. After one hour, E. coli lipopolysaccharide (5 mg/kg) was injected intraperitoneally and 3 hours thereafter blood samples were collected. The number of platelets in the circulating blood were measured to determine the ratio of platelet numbers at pre- and post-administration of the agents. The tests were twice repeated. The result is shown in Table 2. A marked decrease in platelet numbers is found in the control group. Oral administration of 100mg/kg of the compound used in the present invention inhibits the decrease in platelet numbers, which is comparable to an oral dose of 200 mg/kg of ticlopidine.

Test example 2

Effect against disseminated intravascular coagulation:

The agents to be tested were administered orally to male Wistar rats, 7 animals in a group, in an amount of 100 mg/kg. After one hour, E. coli lipopolysaccharide (50 mg/kg) was continuously injected intravenously for 4 hours. The control group was given physiological saline intravenously under the same conditions.
Blood was collected immediately after finishing the injection to measure the number of platelets, activated partial thromboplastin time, prothrombin time and amount of fibrinogen. The result is shown in Table 3. In the control group, to which E. coli lipopolysaccharide was injected, marked decreases in the numbers of platelets and amounts of fibrinogen and prolongation of activated partial thromboplastin time and prothrombin time were observed. In the model of experimental disseminated intravascular coagulation , the compound used in the present invention shows significant effect against all factors tested.

Test example 3

Vasodilating activity:

(1) Effect at an intraarterial administration:

Mongrel dogs anesthetized with urethane ( 450 mg/kg iv ) and α-chloralose ( 45 mg/kg iv ) after pretreatment with morphine (1.5 mg/kg, sc) were held in a supine position. Right femoral artery blood was introduced into the left femoral artery through a circulation pump and the left hind-leg was perfused under constant pressure by connecting with Starling's resistance in an exosomatic circulatory system. The prefusion pressure was set slightly higher than the mean blood pressure of each animal and the change in blood flow by intraarterial injection of the test compound was determined.
The result showing specific activity as compared with a value indicating the degree of increase in the amount of blood flow by papaverine ( 30 µg,iv ) as 100% is shown in Table 4. The compound used in the present invention shows distinctly stronger vasodilating activity than that of the control drug, tetramethylpyrazine.

(2) Effect at an intravenous administration:

A cuff type probe for measuring blood flow was set in a left vertebral artery, left anterior descending coronary artery, left renal artery and right femoral artery of a mongrel dog anesthetized as above. The blood flow was continuously measured. The blood pressure was monitored through a pressure transducer connected to a cannula inserted in the left femoral artery, and the heart rate was monitored through a tachometer by triggering R-wave of lead II electrocardiogram. The agent to be tested was injected into the left venous cephalica. The result is shown in Fig. 1.
In the figure the increasing effect on blood flow of the compound used in the present invention in dogs is shown. The ordinate indicates the response ratio and the abscissa indicates the intravenous administration dose. The following symbols are used:
●: vertebral arterial blood flow;
▲: coronary arterial blood flow;
■: femoral arterial blood flow;
□: renal arterial blood flow;
ο: mean blood pressure;
△: heart rate.
The compound used in the present invention does not affect the renal arterial blood flow and increases the blood flow of vertebral artery, coronary artery and femoral artery. Especially selective effects on the vertebral arterial blood flow and coronary arterial blood flow are observed and so the compound of the present invention is useful for the improvement of brain and heart haemodynamic disorders.

Test Example 4

A protective effect against anoxia:

Male ddY strain mice, 12 animals in a group, were put into an observation chamber filled with 100% carbon dioxide gas. The survival time was observed by signs of apnea. Agents were administered orally 30 or 60 minutes before the test. The result is shown in Table 5.
A significant prolongation in the survival time is observed with any pretreatment of the compound used in the present invention.

Test example 5

Effect against platelet activating factor (PAF) induced shock:

A compound used in the present invention was administered orally to male ddY strain mice, 10 animals in a group, in an amount of 100 mg/kg. After one hour PAF (60 µg/kg) was administered intravenously and the mortality ratio was determined. The result is shown in Table 6 in which shows the protective action of the compound used in the present invention on PAF-induced shock.

Test example 6

Acute toxicity:

A compound used in the present invention was administered to male ddy mice and male Wister rats to test acute toxicity. The fifty per cent lethal dose was approximately 200 mg/kg in mice. No deaths were observed at an intravenous dose of 100 mg/kg in mice. Rats can only be administered with up to 50 mg/kg of the compound due to the solubility of the compound. No deaths were observed at the dose of 50 mg/kg intravenously. Fifty per cent lethal oral doses were 555 mg/kg for mice and 903 mg/kg for rats.

Test example 7

Cytotoxicity:

To L5178Y murine lymphoblastoma ( 2 x 10⁴ cells/mℓ) suspended in a medium (27 ml) comprising Fischer's culture medium and containing 10% bovine serum was added test agent solution (0.3 ml) diluted with culture medium. The mixture was incubated at 37 °C for 18 hours. The growth of cells was determined by color change. Growth inhibition of cells was not observed at concentrations of 20 µg/ml and 100 µg/ml of the compound used in the present invention. Thus no cytotoxicity was observed.

Test example 8

Mutagenicity:

Backward mutation test using microorganisms strains TA100, TA1535, WP2 uvr A, TA98, TA1537 and TA1538 was performed by a method according to Iyer et al. [N.Y.Iyer and W. Syzbalski,Appl. Microbiol., 6: 27 (1958)]. Test agents were diluted at 6 step concentrations of 0.00333 ∼ 333 µg/disc at dilution ratio of 10:1. No mutagenesis was found at any concentration of the compound used in the present invention. Also no mutagenesis was observed under metabolic activation conditions using a S-9 mixture.
As clearly illustrated, 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or non-toxic salt thereof has an inhibitory effect on platelet aggregation, vasodilating activity and protective effect against anoxia with low toxicity. It can be used to improve haemodynamic and metabolic disorders.
The compound used in the present invention can be prescribed per se or in various forms of administration orally or parenterally, such as intramuscularly, subcutaneously, intravenously, rectally or cutaneously. The preferred oral daily dose is from 1 to 600 mg for one man. The composition comprising the compound used in the present invention can be in solid form, such as a tablet, sugar coated tablet, film tablet, hard or soft capsule, trouch, pill, granules and powder, semi-solid form such as a suppository, cataplasm and ointment, or liquid form such as an injection, syrup, inhalant, emulsion and suspension. The compound can be used in the above formulations per se or can be mixed with other components.
The following Examples further illustrate the present invention

Example 1

Injection

2-hydroxy-3-isopropyl-5,6-dimethylpyrazine hydrochloride (10 mg) was dissolved in distilled water for injection (20 ml), adjusted to a concentration of 0.5 mg/ml, sterilized, and filled up to 1 ml in an ampule to prepare the injection.

Example 2

Tablets (200 mg/tablet) comprising the above composition were prepared by a dry tabletting method.

Example 3

Drip Infusion:

Injectable liquid formulae containing 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine hydrochloride (10 mg) was diluted in 5% glucose solution (250 ml) to prepare a drip infusion.

Claims

A process for the preparation of a pharmaceutical composition, which process comprises mixing 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine or a non-toxic salt thereof with a pharmaceutically acceptable carrier or diluent, the said mixing being effected under sterile conditions and/or the obtained mixture being sterilised.
A process according to Claim 1 wherein the salt is the hydrochloride.
A process for the preparation of a non-toxic salt of 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine, which process comprises condensing 2,3-butanedione with valineamide in the presence of a tertiary amine and converting the resultant 2-hydroxy-3-isopropyl-5,6-dimethylpyrazine into a non-toxic salt thereof.
A process according to Claim 3 wherein the salt is the hydrochloride.